P1423: CIRCULATING CELL-FREE DNA KINETICS MEASURED BY DIGITAL PCR IN LYMPHOMA PATIENTS UNDERGOING CD19-CAR-T THERAPY
Authors: M. López-Esteban, D. Carbonell, M. Bastos-Oreiro, I. de la Iglesia, A. Pérez-Corral, M. Chicano, P. Muñiz, L. Sanz-Villanueva, R. Bailén, G. Oarbeascoa, J. Anguita, M. Kwon, J. L. Díez-Martín, I. Buño, C. Martínez-Laperche
Journal: HemaSphere (2022)

Background: Monitoring of CD19-targeted chimeric antigen receptor (CAR)-T cell levels is essential for an optimal follow-up of patients. In this regard, methodologies such as multiparametric flow cytometry (MFC) or qPCR are commonly performed. In addition, the use of new approaches, such as digital PCR (dPCR), has improved the analysis of cell kinetics. Nevertheless, the role of CAR circulating cell-free DNA (ccfDNA) dynamics during CAR-T cell treatment is not clear. Aims: To evaluate the usefulness of CAR ccfDNA measurement by dPCR in patients treated with CD19-CAR-T cell therapy and its correlation with CAR-T cell levels measured by MFC and dPCR. Methods: Forty patients diagnosed with a B-cell lymphoma who underwent CAR-T therapy (28 Axicabtagen Ciloleucel and 12 Tisagenlecleucel) between May 2019 and May 2021 were included in the analysis. One hundred and five peripheral blood (PB) samples were collected in EDTA tubes at days +1, +7, +14, +30 (10, 31, 33, and 31 samples, respectively) after CAR-T administration. Plasma was obtained from 20 mL of PB by centrifugation at 2500g for 30 minutes at 4°C. DNA was purified from PB samples using the Maxwell 16 Blood DNA Purification Kit (Promega) and ccfDNA was extracted from 5 mL of plasma, using QIAamp® Circulating Nucleic Acid (Qiagen). dPCR was carried out in DNA and ccfDNA samples, using specific primers and probes for FMC63 and a reference gene. MFC was performed on a DxFLEX cytometer (Beckman Coulter), using CD19 (20-291) protein-FITC (ACRO Biosystems). Continuous variables were expressed as median and range. Correlations between CAR copies in plasma determined by dPCR and CAR-T cells measured in PB by MFC and dPCR were analyzed using Pearson’s test. Comparison between number of copies and clinical variables (cytokine release syndrome -CRS-, immune effector-cell associated neurotoxicity syndrome -ICANS-, and relapse) was analyzed using Mann-Whitney U test. All statistical analyses were performed using GraphPad Prism 8.0.1. Results: Median values of CAR copies of ccfDNA per milliliter measured by dPCR were 39, 38, 25 and 2 at days +1, +7, +14 and +30. Similarly, median reference gene values were 3079, 1799, 2588 and 2183 (Figure 1). Thus, we validate previous observations of an initial peak concentration followed by a constant decrease in plasma CAR copies kinetics. Moderate correlation was found between CAR copies/mL of plasma and CAR-T cells in PB determined by MFC and dPCR (R=0.49, p<0.001, Figure 2A; R=0.51, p<0.001, Figure 2B, respectively). So, although a part of plasma CAR copies variation is explained by changes in whole blood CAR content, there might be other influencing factors. Regarding the association between CAR copies in plasma at +7 and clinical variables, an association was only found in the case of development of CRS<2 and CRS grade ≥2 (28 [0-220] vs 168 [11-1479], p<0.01, Figure 3). Further experiments are needed to evaluate whether this reflects the overall CAR-T cell kinetics. Image:Summary/Conclusion: The analysis of multiple variables is important for an optimal monitoring of patients undergoing CAR-T cell therapy. In this sense, ccfDNA dynamics may play an important role during the follow-up and even have relevant clinical implications as we have demonstrated in the present study. However, more studies are necessary for validating our data.